A SILAC-based DNA protein interaction screen that identifies candidate binding proteins to functional DNA elements

Abstract

Determining the underlying logic that governs the networks of gene expression in higher eukaryotes is an important task in the post-genome era. Sequence-specific transcription factors (TFs) that can read the genetic regulatory information and proteins that interpret the information provided by CpG methylation are crucial components of the system that controls the transcription of protein-coding genes by RNA polymerase II. We have previously described Stable Isotope Labeling by Amino acids in Cell culture (SILAC) for the quantitative comparison of proteomes and the determination of proteinprotein interactions. Here, we report a generic and scalable strategy to uncover such DNA protein interactions by SILAC that uses a fast and simple one-step affinity capture of TFs from crude nuclear extracts. Employing mutated or nonmethylated control oligonucleotides, specific TFs binding to their wild-type or methyl-CpG bait are distinguished from the vast excess of copurifying background proteins by their peptide isotope ratios that are determined by mass spectrometry. Our proof of principle screen identifies several proteins that have not been previously reported to be present on the fully methylated CpG island upstream of the human metastasis associated 1 family, member 2 gene promoter. The approach is robust, sensitive, and specific and offers the potential for high-throughput determination of TF binding profiles.

The Role of WRKY Transcription Factors in Plant Immunity

“…Nevertheless, to fully understand regulation, we need to gain access to the full set of proteins associated with WRKY TFs at specific genomic loci. Indeed, promising technological advances combining DNA probes and mass spectrometry, such as proteomics of isolated chromatin segments and stable isotope labeling with amino acids, are starting to demonstrate that identification of TFs and associated proteins in vivo at given promoters may become feasible in the near future (Dèjardin and Kingston, 2009;Mittler et al, 2009).…”

Quantifying the effect of sequence variation on regulatory interactions

“…Clearly, much work remains to be done. However, there is hope that the theoretical developments will increasingly be driven by technological advances and quantitative data [Mittler et al, 2009], against which the models can be optimized.…”

DNA-centered approaches to characterize regulatory protein–DNA interaction complexes

“…Recent examples of the DNA affinity chromatography approach include the isolation of a Drosophila TF, DEAF-1, binding to the enhancer of an immunity gene, 26 as well as several proteins binding to promoter fragments of, respectively, the human ESRRA and MTA2 genes. 27 In both studies, DNA was immobilized onto a solid phase by biotin-labelling the DNA and coupling it to either streptavidin-coated columns or magnetic beads. This is in contrast with the technique of DNA trapping used by Jiang and co-workers 29 in which a 250 bp region of the human c-jun promoter with a single stranded (GT) 5 tail was annealed to single-stranded (AC) 5 -Sepharose.…”

“…35 The second is the Orbitrap, which also uses an FT-based strategy. 36 For example, Mann and co-workers 27 have used an FT-ICR to identify sequence-specific DNA-binding proteins in HeLa S3 cells purified by DNA affinity chromatography. Interestingly, when comparing the eluted protein SDS-PAGE profiles from the wild-type and negative control DNA bait, there was virtually no difference and thus no clear bands were revealed corresponding to true specific DNA-binding proteins.…”